Take a look at your reflection in a mirror. If you’re impressed by what you see, you should be. Everything about you, everything you can and cannot see, is maintained by a complex network of biochemical pathways in your cells. Every year as scientists seek to understand the specifics of genetics and cell biology, they decode small bits of this puzzle, and work their way closer to solving the mysteries of why diseases arise and how they can be stopped. The discovery of RNA interference (RNAi) by Andrew Fire at Stanford University and Craig Mello at the University of Massachusetts, a discovery that garnered them a Nobel Prize in Medicine in 2006, represented a huge breakthrough in the quest to figure out how gene expression is regulated, a key step in controlling the expression of wayward or disease-causing genes. The wake of RNAi’s discovery has manifested broadly in academia and industry alike, and has generated hopes for therapeutics that could be precisely targeted to individual misbehaving genes. The field of RNAi therapy has burgeoned in the past decade; however, in some cases expectations have outpaced the tumultuous path of discovery. Almost fifteen years after the first publication delineating the mechanisms behind RNAi, some researchers have abandoned the field altogether. And yet, some believe it is uniquely poised to deliver important results. Looking at the state of the field, graduate students and post-docs in Fenyong Liu’s laboratory in the UC Berkeley School of Public Health are part of a larger movement in RNAi therapeutics—but they could be taking a great risk on an uncertain therapy.
RNAi is a ubiquitous process by which the expression of genes is regulated by small bits of double-stranded RNA made by the cell. According to the central dogma of molecular biology, DNA is transcribed into messenger RNA that ultimately encodes a protein, but it’s often not that simple in practice. RNA interference is one of a multitude of regulatory mechanisms that can step in between a genetic recipe and its intended protein output. Perhaps one of the most exciting uses for RNA interference is therapeutics. By using the blueprints for natural RNA interference, biologists are able to synthesize components of the RNAi pathway that can repress the expression of genes in diseased cells.
Scientists throughout the field of molecular biology have been hard at work since the discovery of RNA interference, attempting to turn the idea of RNAi therapeutics into a reality. RNAi therapeutics has been shown to work in in vitro (cellular) models in the controlled conditions of a lab. However, any RNAi-based drug faces a slew of unforeseeable variables when introduced into a living system. One major barrier to the success of RNAi therapeutics in live organisms is the mode of delivery. Taking a pill is too simple; RNAi would be quickly degraded by the digestive system. An injection could trigger degradation by an immune response, and wouldn’t be specifically targeted to diseased cells. Currently, there is no safe and effective way of getting RNAi therapeutics through our bodies and into cells. This might seem to be a game-ending proclamation, but with the potential benefits of RNAi therapeutics in mind, the prospect of engineering a novel delivery mechanism keeps scientists hard at work. Scientists in the Liu lab are working to design effective delivery mechanisms for RNAi therapeutics that work with the body’s natural systems, thus reducing toxicity and negative side effects. They are engineering guide sequences that would direct RNAi to specific cells, and using gutted and harmless bacteria as delivery vehicles.
There are factions of RNA interference: microRNA (miRNA) and small interfering RNA (siRNA). The main difference between the two is their origin: miRNAs are endogenous, or natural; siRNAs are generally exogenous and are synthesized in a lab. siRNAs are modeled after naturally occurring miRNA. They both begin as long, double-stranded fragments of RNA, and are then processed by a series of enzymes into short fragments of just 22-27 nucleotides. These fragments can pair with complementary segments of mature cellular messenger RNA—the RNA that codes for proteins—and, with the help of an enzyme complex, cleave it. RNAi cleavage marks specific strands of messenger RNA for destruction by the cell’s natural machinery, before they can be translated into protein precursors.
RNA interference therapy: re-working an old standby
The real fascination with RNAi lies in the prospects for its use as a therapeutic for gene-based diseases. The idea is to engineer siRNAs (synthetic double-stranded RNA fragments that are about 22-27 nucleotides in length) that specifically target genes within certain cells that are associated with disease or that are necessary for replication. The cells that are targeted have been functionally taken over by a genome, or genetic mutations, that cause them to make proteins that disrupt a cell’s harmonious relationship with its neighbor. This happens with cancerous cells and cells that have been infected by a virus. In the case of cancer, genes that promote mitosis (cell division) are expressed at levels that are far higher than normal. RNA interference therapy could knock down the expression of those genes. In the case of viruses that take over their host’s cellular machinery and induce the expression of their own viral components, RNA interference therapy could be engineered to target the virus’s own genes, hindering its ability to reproduce. The beauty of RNA interference therapy lies in its origin as a naturally occurring process in cells.
Theoretically, therapy would involve synthesizing double-stranded siRNA in a laboratory and introducing it into a target cell. This synthetic RNA would pair with target messenger RNA. The siRNA would then proceed through the normal RNAi pathway. By waylaying the genetic code in its quest to become a protein, the RNA interference pathway can knock down the expression of a specific gene.
If we can agree that the theory sounds promising, then where are the results? There are a number of competing therapies for cancer on the market, including chemotherapy, radiation, and monoclonal antibody drugs (like Avastin), all widely administered; where do RNAi therapeutics fit in? Why hasn’t this nearly magical technique changed the way we treat disease? RNAi isn’t used to treat disease in humans—yet. RNAi has been shown to work in many in vitro and some in vivo (animal) experimental models, but development of therapeutics for human use is a slow process for even the most successful methods. This isn’t for lack of trying; along with researchers in academia, many biotech companies have RNAi divisions, and even subcompanies. Merck, for one, invested $1.1 billion in 2006 to acquire Sirna Therapeutics, a company that, true to its name, is investigating siRNA treatments. Roche has partnered with Alnylam, another company researching siRNA based in Massachusetts, and smaller startups like Regulus Therapeutics are operating in the same field. Yet every company being funneled billions of dollars of research funding has come up against the same obstacle: delivery.
In order to produce a viable and marketable therapeutic, scientists must not only find a way to direct RNAi drugs across the cell membrane and to their target tissues, they must also circumvent the body’s enzymatic pathways. Normally, the body would break down a free double-stranded RNA before it could have any effect on a cell. If the double-stranded RNA reaches a cell, passing it through the cell membrane becomes a difficulty. The cell is very selective in what it allows into its cytoplasm, and foreign or synthetic substances are highly restricted. The goal, then, is to disguise RNAi therapeutics as natural components of our bodies.
Here’s where UC Berkeley scientists could make an enormous impact. Dr. Naresh Sunkara, a postdoc in Liu’s lab, works with delivery mechanisms for RNAi therapy. He believes that by constructing a molecule that mimics some of the body’s natural chemistry, he will be able to walk that fine line between getting drugs past the cell’s membranous barrier and avoiding activating an immune response. Sunkara is investigating using synthetic lipids, which can mimic the natural composition of the cell membrane, to contain and deliver therapeutic siRNA. Lipids interface well with cell membranes, and form protective structures that can shield siRNA from enzymes or immune responses until they reach cells. However, targeting can be a problem; lipids primarily end up in the liver, whereas cancerous cells can reside in any part of the body.
Another obstacle for siRNA therapeutics is off-target effects. Despite the specificity of siRNA engineered to bind only one particular segment of a gene, biology has a way of providing unintended consequences. If an siRNA molecule is complementary to part of a gene, rather than the entire transcript, it can still function to block transcription of that gene, if only partially. This means that siRNA has to be designed extremely carefully, so that it not only affects the target gene, but also doesn’t match up, even in part, with genes that code for other proteins not being targeted. Off-target effects can also involve the natural RNA machinery that siRNA takes advantage of for processing. An overabundance of introduced siRNA molecules can mean that the naturally-occurring mRNA meant to be processed by the cell isn’t taken care of efficiently, or isn’t processed at all. This siRNA saturation can be toxic to a cell, as it hinders any RNA, including functionally necessary protein-encoding mRNA, from leaving the nucleus and being translated. Off-target effects can be screened for in animal models, but the cost of their unpredictability can be a deterrent to development.
The broader goal of Sunkara and others who work to engineer cancer therapeutics using RNAi is potentially market-changing: the hope is to phase out the use of chemotherapeutic and non-specific drugs. Sunkara explained that the cause of the adverse side effects and failures of chemotherapy lie in the fact that we all have unique genotypes, which a general course of action like chemo is not equipped to address. “The best course of action is to understand our biology better, and tailor our drugs based on our personal DNA or RNA,” Sunkara explained.Initiatives like The Human Genome Project opened the door for huge advances in our ability to understand the specific functions of our genes and marked the beginning of an explosion in gene therapy research. As we gain a firmer grasp on our own biology, the possibilities for gene-specific therapeutics open up. “This is where RNA interference comes in; many diseases that we see involve the up-regulation of certain genes,” said Sunkara.
Sunkara believes that the future of pharmaceuticals lies in identifying deranged genes and regulating them through mechanisms like RNA interference. He painted a picture: “Suppose you have the flu. You go to a hospital, they take your blood, genotype your cells to see what exactly is wrong, and then actually design something that will correct the problem. Then you come back the next day to get a drug specific to your problem.” In terms of cancer treatment, he sees great potential for the activity of genotype-specific drugs. “The genotypes in cancer cells are bound to be different in different human beings,” he said. RNA interference therapy, with its intrinsically specific binding properties, offers us greater control over disease management.
For existing cancer drugs, general effectiveness is a valuable quality. Drugs like those used in chemotherapy can be administered to a wide range of patients, and are deemed efficient; they have the potential to provide answers to many ailments. However, when general drugs do not work, Sunkara believes that specific treatments, while labor-intensive and still mainly theoretical, will help healthcare professionals work with our varied genotypes to circumvent side effects and ineffective treatments.
Discussing the future of RNAi therapeutics, Sunkara also noted the difficulties that the business of drug development can present. He pointed to the fairly recent cutbacks that Roche Pharmaceuticals, the world’s biggest funder of drug research and development (R&D) implemented. After planning on investing $1 billion in RNAi R&D, Roche completely shut down its RNAi division. Novartis, another premier pharmaceutical company, also shut down its RNAi operations. The corporate decision-making behind this high-profile exit from RNAi therapy is rarely disclosed. In the private sector of drug development, scientific pursuits are more heavily exposed to market demands than are governmental or academic projects. Indeed, scientists like Sunkara see these shutdowns as products of market forces rather than a reflection of RNAi therapy’s viability. He acknowledges the setback that RNAi therapy has suffered because of a lack of support from the private sector, but seems undaunted on the whole.
Dr. Paul Rider, also a post-doc in the Liu lab, pointed out that although we may not see RNAi therapeutics being administered to patients with cancer or viral infections, there have been some successes in developing therapeutics that have moved on to phase I and II clinical trials. Indeed, one of the reasons that we do not see RNAi drugs on the market today may be that it is such a young idea. Monoclonal antibodies went through a similar period of hype in the 1980s, followed by a longer-than-expected developmental period before the first viable products came to market. Drugs must pass through four trial phases in order to ensure safety, which can take more than a decade. Concerns with delivery methods could slow passage through these trials even further.
Regardless of the challenges, many scientists still believe that these sequence-specific drugs are the wave of the future. Undoubtedly, as our understanding of the specific aberrations in cell biology that lead to disease continues to expand, we will see an increase in drug specificity. Despite the inherent barriers that RNAi therapy presents, it offers the tantalizing possibility of drug specificity.
Humans are innately fascinated by our origins, the mystery behind life, and the idea that we can improve the conditions of life within our own lifespans. We are increasingly developing the potential to uncover causes and treatments for cancer, viral and bacterial infections, inherited genetic disorders, and even addiction. Even as they persevere to account for all the variables of the human body and its microenvironments, biologists like Sunkara cannot help but be optimistic about the prospects for RNA interference therapy and specialized medicine. It may take longer than expected to see this research investment pay off, but scientists at UC Berkeley are no strangers to high-risk, high-reward projects.